Physicochemical Differences between Ascitic
and Solid Forms of Sarcoma 37 Cells
G. M. W. COOK,G. V. F. SEAMAN,*ANDL. WEISS
(Department of Radiotherapeutics, University of Cambridge; and Medical Research
Council, Strangeways Research Laboratory, Cambridge, England)
SUMMARY
It was shown by cell electrophoresis that there was a significant difference in mo
bility between cells derived from the ascites and solid forms of murine Sarcoma 37.
Treatment
of cells from both forms with neuraminidase
showed a highly significant
reduction in mobility for the ascites type, whereas no measurable change in mobility
occurred for the solid type. Sialic acids were released from both the ascites and solid
forms of cell by the action of neuraminidase.
The evidence presented suggests that the
reversible transformation
from ascites to solid form is a modulation,
and we have
measured some of the physicochemical differences associated with this type of reversi
ble transformation.
If ascites Sarcoma 37 cells are injected into the
peritoneal cavities of mice, most of the tumor cells
may be harvested as a single-cell suspension in the
peritoneal fluid. On the other hand, cells injected
subcutaneously
form a discrete solid mass of mu
tually adherent cells. If cells from the same pool
are injected intraperitoneally
and subcutaneously
into mice of the same strain and differences are
observed between cells subsequently
taken from
the two sites, these differences arise from the in
teraction of the cells with their environment.
This communication
is concerned with an at
tempt to detect some physical and chemical differ
ences between the solid and ascitic forms of
Sarcoma 37, 7 days after implantation. Sialic acids
are important constituents
of many animal cells,
contributing
to their electrokinetic
charge and
possibly their adhesive properties. It was decided
therefore to examine the electrokinetic properties
of the cells and the modification of these proper
ties by treatment
with neuraminidase,
coupled
with a microchemical
examination
of the sialic
acids released by the action of this enzyme.
MATERIALS
AND METHODS
Chemicals and solvents were of AnalaR grade,
and procedures were carried out at room tempera* Present address: Division of Neurology, University of
Oregon Medical School, Portland 1, Oregon.
Received for publication July 8, 1963.
ture (ca. 20°C.) unless otherwise stated. The water
used was distilled twice in Pyrex ware, and all
systems were in equilibrium with the atmosphere.
Freshly harvested ascitic fluid (0.3 ml.) from
tumor-bearing
animals was injected either subcu
taneously (right flank) or intraperitoneally
into
young, adult male mice of the CBA strain (No.
797b, Laboratory Animals Centre Catalogue of
Uniform Strains,
2d edition).
After 7 days,
growth appears to be maximal as the solid tumors
begin to exhibit central necrosis, and the ascites
tumors also reach a stationary phase of growth.
Seven days after tumor injections mice were
killed by cervical dislocation, and the ascitic fluid
was removed. If the cells were not to be immedi
ately separated from the suspending fluid by centrifugation,
1 volume of ascitic fluid was mixed
with 0.05 volumes of 3 per cent w/v EDTA disodium salt in 0.7 per cent w/v aqueous NaCl. The
dissected solid tumors were kept moist with this
EDTA solution.
The ascites tumor cells were freed from the
ascitic fluid either by gradual dilution of this fluid
with approximately
15 volumes of physiological
saline (0.145 M NaCl:3 X 10~4 M NaHCO3, pH
7.2 + 0.2) or by immediate removal of the undi
luted ascitic fluid after centrifugation.
Both cell
suspensions were washed 3 times with 15 volumes
of physiological saline to 1 volume of packed cells
(2 min., 500 X g).
1813
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1814
Cancer Research
The solid tumors were minced between two
scalpels, and the mince was briskly pipetted back
ward and forward for a few minutes. After the
larger pieces of tumor had settled out a tumor cell
suspension was obtained, which provided enough
cells for re-injection and examination by cell electrophoresis.
When larger quantities
of cellular
material were required, solid tumors were put
through a modified Craigie mincer (5). Solid tu
mors were placed in a stainless steel tube sealed
at one end, and their constituent cells were sepa
rated by forcing them past a stainless steel plunger,
bearing V-shaped left- and right-handed
spiral
grooves (2 threads per inch) having a depth of
0.015 inches (0.38 mm.) and a width of 0.02 inches
(0.51 mm.). The isolated tumor cells were washed
three times in physiological saline in a manner
similar to that for the ascites tumor cells.
Treatment with neuraminidase.—After
the cells
(ascites and solid) were washed with physiological
saline they were washed once in calcium-saline
(0.145MNaCl:0.005MCaCl2:3
X 10-4MNaHCO3,
pH 7.2 ±0.2). Equal volumes of the packed (2
min., 500 X g) tumor cells were then treated with
either the same volume of neuraminidase solution
(1 volume of stock Behringwerke neuraminidase
of activity 100 units1 per ml. to 4 volumes of cal
cium-saline) or, in the case of controls, with 0.8
of their volume of calcium-saline.
Although ini
tially the period of incubation at 37°C. was 60
minutes, since it was later found that changes in
the electrophoretic
mobility were complete after
30 minutes, the incubation time was consequently
reduced to this to minimize cellular damage. After
incubation
the cells were centrifuged
(10 min.,
500 X g), and the supernatant
fluids were re
moved. The supernatants from the control systems
were treated with 0.2 of their volume of neura
minidase solution and incubated for a further 30
minutes. All supernatant fluids were then analyzed
microchemically.
The tumor cells which had been treated with
neuraminidase
were washed twice in physiological
saline and examined by cell electrophoresis.
Treatment of cells from the solid tumor with cellfree ascitic fluid.—One volume of packed cells
(solid tumor) was incubated with an equal volume
of cell-free ascitic fluid for 30 min. at 37°C. Ascitic
fluid with and without added EDTA was used in
these experiments,
and the treated cells were
1 One unit of neuraminidase
activity will release 1 ng. of
N-acetylneuraminic
acid in 15 minutes at 37°C. from a suit
able glycopeptide substrate dissolved in 0.05 M sodium acetateacetic acid buffer at pH 5.5 made 0.9 per cent with respect to
sodium chloride and 0.1 per cent with respect to calcium
chloride.
Vol. 23, December
1963
washed twice in physiological saline. Neuramini
dase treatment of the tumor cells which had been
previously incubated with ascitic fluid was carried
out as described in the previous section.
Microanalyses.—Supernatant
fluids from the
above controlled neuraminidase
experiments were
deproteinized by adding an equal volume of aque
ous trichloroacetic
acid solution (TCA) (10 per
cent w/v). After flocculation and centrifugation
(2 min., 500 X fir), portions (0.2 ml.) of the clear
supernatant
were assayed for sialic acids (results
expressed as the N-acetyl derivative [NANA]) by
Warren's technic (21). Ascitic fluid after acid
treatment (0.1 N H2SO4 at 80°-90°
C. for 1 hour)
was also assayed for sialic acids.
Packed ascites cells and minced solid tumors
which had been washed in saline were extracted
with chloroform/methanol
(2:1 by volume) using
the procedure of Dawson et al. (6). After filtration
and removal of the solvent system by distillation
under reduced pressure, white waxy residues were
obtained. Insufficient material was isolated for the
total sialic acid content of these 'crude lipide'
fractions to be estimated by the methanolic HC1
technic (27). Since unsaturated
lipides may yield
malonaldehyde
(2) upon oxidation with periodate,
attempts to determine the presence of sialic acids
in this material by the Warren technic (21) were
abandoned.
Characterization of sialic acid present in solid
tumor.—About 80 tumors were dissected from 40
mice and minced (5) in physiological saline, and on
centrifugation
(5 min., 500 X g) 16 ce. of packed
cells was obtained.
A small proportion
of the
cells was removed for electrophoretic examination
while the remainder was washed (washing ratio,
1:1) once in twice-distilled water followed by two
washes in acetone and one wash in 0.05 N sulfuric
acid. After this procedure 2.3 cc. of packed (5
min., 500 X g) material was obtained and was
suspended in 0.05 N sulfuric acid (7 ml.) and left
at 80°-90°
C. for 1 hour. After cooling and centrif
ugation the supernatant
fluid was deproteinized
with an equal volume of trichloroacetic acid solu
tion (10 per cent w/v). Centrifugation
followed
by extraction (X5) with diethyl ether yielded a
clear supernatant
whose pH was brought to 6.0
with saturated barium hydroxide solution. Follow
ing further centrifugation,
to remove the barium
sulfate precipitate, the clear fluid was assayed by
the Warren method; a correction to the optical
density of the chromophore at 562 m p by reference
to the optical density at 532 m p was made as
suggested by Warren (21) and utilized by Wallach
and Eylar (20) for acid-treated cell homogenates.
Sialic acid (NANA) (100-150 /¿g.)was shown
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COOK et al.—Electrophoresis
to be present in this fluid. After freeze-drying,
the lyophilized material was chromatographed (3)
alongside authentic NANA with butan-2-olracetone: acetic acid:water (30:30:15:25) by volume
in a descending direction on acid-washed What
man No. 1 paper. Trace quantities of N-acetyl
neuraminic acid were identified in the experimen
tal strip following development with orcinol-trichloroacetic acid reagent.
Cell electrophoresis.—Mobility measurements
were made in a modified cylindrical chamber appa
ratus at 25°C. ±0.1°(19). All cells were sus
pended in physiological saline at pH 7.2 + 0.2 for
examination of their electrophoretic properties.
The tumor cell suspensions were examined be
tween 1 and 3 hours after being harvested from
the mouse, and during this period the parameters
measured showed no significant change.
of Sarcoma 37 Cells
1815
formalin or Zenker's fluid, as were solid tumors;
after sectioning they were stained, by standard
methods, with hematoxylin and eosin, periodic
acid-Schiff, Hale's stain, Mallory's trichrome (16),
and Warren and Spicer's stain (22) for sialic acidcontaining mucins. No differences could be de
tected between the solid and ascitic forms of
Sarcoma 37 by these methods.
RESULTS
The electrophoretic mobilities of cells derived
from either the ascitic or solid form of Sarcoma
37, untreated or following treatment with neuraminidase or ascitic fluid, are given in Table 1.
The difference between the mobilities of the un
treated (control) ascites and solid tumor cells was
significant (P<0.001, with 41 degrees of freedom).
Treatment with neuraminidase produced a 30-35
TABLE 1
ELECTROPHORETIC
MOBILITIESOFNORMAL,ASCITICFLUIDANDNEURAMINIDASETREATEDSARCOMA
37 CELLSANDSlALICACIDRELEASEDBYNEURAMINIDASE
ur ACID
RELEASED BY
NEURAMINIDASE(MO
Cll)Control-l.16iO.12-0.9210.08Neuraminidase0.77±0.10
(it SEC"1 v"1
TYPE OF
CELLAscitic
TUMOR
fluidNo
form
Solid formMOBILITY
NANA/cc
PACKED
CELLS)15-20*
change from
valueNo
control
0.94 + 0.09Ascitic
change from
control valueSit
* Removal of ascitic fluid by gradual dilution,
t Ascitic fluid removed prior to saline washing.
Control mobilities for the cells obtained from
the solid tumors by either chopping or mincing
were identical. The use of EDTA as an anticoagu
lant in the ascitic fluid and its addition to the
solid tumor cell suspension produced no significant
change in the electrophoretic mobility of the cells.
Viability tests.—Cellviability was assessed both
after washing and standard treatment with neura
minidase by appearance under the phase-contrast
microscope and by trypan blue uptake test (15).
These tests have previously been correlated with
animal inoculation experiments (23). After both
treatments about 90 per cent of the sarcoma cells
were viable. Approximately 90 per cent of the
total cell population consisted of sarcoma cells,
and the remainder were macrophages and leuko
cytes. No erythrocytes or bacteria were seen in any
sample.
Histochemistry.—Ascites tumor cells were centrifuged into plugs which were fixed in neutral
per cent reduction in the mobility of cells in the
ascitic form. Analysis by the Student's "t" test
showed that this reduction was highly significant
(P «0.001, with 33 degrees of freedom). On the
other hand, the difference in mobility between the
control and neuraminidase-treated
solid tumor
cells was not significant (P ~ 0.4, with 34 degrees
of freedom).
Cells derived from the solid tumor, which had
been previously incubated with ascitic fluid, in the
presence or absence of EDTA, showed no reduc
tion in the electrophoretic mobility after neura
minidase treatment. After 7 days' growth some
solid tumors were minced between scalpels and
injected into the peritoneal cavities of fresh mice.
The resulting ascites cells were harvested and ex
amined by electrophoresis before and after neura
minidase treatment, and compared with ascites
cells which had been maintained continuously in
the ascitic form. No electrokinetic differences
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1816
Cancer Research
could be detected between the two batches of
ascites cells which were both different from the
solid form.
A series of analyses on ascitic and solid tumors
revealed no significant difference in the amounts
of sialic acids released (Table 1). Large quantities
of sialic acids (100-300 pg/ml, NANA) were dem
onstrated in the ascitic fluid itself, indicating the
necessity of thoroughly washing the ascites cells
before analysis, to avoid adsorption artefacts.
DISCUSSION
The present findings show reversible physicochemical differences between two cell types de
rived from a common pool.
Only those charge groups which are located
within about 10 A of the hydrodynamic slip plane
of a cell will contribute to its electrophoretic char
acteristics in physiological saline (9). The magni
tude of the electrophoretic mobility is an indica
tion of the net electrokinetic charge but not of the
nature of the ionogenic groups or of the uniformity
of their distribution throughout the electrokinetically effective zone (25).
Cells from the solid tumors possess significantly
lower electrophoretic mobilities than do the ascites
tumor cells (Table 1). In these experiments we
have compared certain properties of cells from
7-day ascites tumors with 7-day solid tumors. It
is well known that, owing to different environ
mental conditions, the initial growth rate of cells
within the peritoneal cavity is higher than that in
a subcutaneous site. Recent work (7, 10, 18) has
drawn attention to the fact that the net negative
charge on cells is often related to their growth rate.
It can, therefore, be seen that there are dangers
in comparing cells having different growth rates
and correlating any differences between them with
biological properties other than growth rate at the
time of the observation. We have attempted to
minimize this difficulty by studying cells in the
steady-state. After 7 days, some of the solid tu
mors showed minimal central necrosis which be
came progressively more marked with time. The
animals with ascites tumors usually died on or
about the 10th day. It appeared, therefore, that
7 days was the maximal time that tumors could
be left in the animals without facing the problems
associated with degeneration. Studies on tumors
left for shorter periods in the animal have not
been carried out yet, owing to the comparatively
large quantities of material required for chemical
analyses (21). It may be mentioned that no elec
trokinetic differences could be demonstrated be
tween 5-, 6-, 7-, 8-, and 9-day Ehrlich ascites tu
Vol. 23, December
1963
mors (Seaman, unpublished data), thereby con
firming the preliminary observations of Ambrose,
James, and Lowick (1). This is perhaps surprising
in view of the suggestion that charge is related to
growth rate (7, 10, 18).
The electrophoretic properties of cells arise
principally from the presence of ionogenic groups
at their peripheries and possibly the redistribution
of ions in the interfacial region. These properties
may be significantly modified by adsorption of ex
tracellular substances, intracellular leakage prod
ucts, and, in the case of neuraminidase treatment,
elimination of sialic acids from the peripheral zone
of the cells. Rearrangement within this region
initiated by elimination of the sialic acids or ad
sorption of the enzyme or of material leaking from
the cell as a result of the enzyme action may also
modify the electrokinetic properties.
In the solid Sarcoma 37 tumor the individual
cells have to be isolated mechanically from the
parent tumor. Differences in electrokinetic prop
erties between the solid and ascitic forms could
arise from inadequate removal of adherent cellular
debris from the separated solid tumor cells (24).
In addition, adsorption of various components
from the environments of the cells during isolation
and washing may also account for the differences.
At least some of the sialic acid associated with
the ascites cells could have arisen from adsorption
of sialo-substances from the ascitic fluid. However,
incubation of cells from the solid tumor with cellfree ascitic fluid lacking or containing EDTA did
not produce any detectable effect on their electro
phoretic mobility. Also, the solid tumor cells incu
bated with ascitic fluid showed no change of
mobility with respect to the control either before
or after treatment with neuraminidase, indicating
that, if adsorption of components from the ascitic
fluid had occurred, as is likely from the work of
Holmberg (11), then such processes are not re
flected in either the electrokinetic charge or neura
minidase susceptibility of these cells.
The reduction in mobility of the ascites tumor
cells following treatment with neuraminidase sug
gests that, as in the case of Ehrlich ascites carci
noma cells (4, 20) and malignant rat liver cells (8),
at least some of the surface charge in the Sarcoma
37 ascites cells arises from the carboxyl group of
a sialic acid. Analyses of the supernatant fluids
from the neuraminidase-treated
cells indicates
that sialic acids are released from both the ascitic
and solid tumor cells, although no change in
charge occurs for cells obtained from the solid
tumors. It is probable that the sialic acid mole
cules are released from different positions within
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COOK et al.—Electrophoresis of Sarcoma 37 Cells
the peripheral zone, so that some molecules con
tribute only partially, if at all, to the electrokinetic
properties of the cells (4, 20). It has also been sug
gested (8) that the carboxyl group of the sialic
acid may not be free, so that the elimination of
such molecules would not affect the electrokinetic
properties of the cell. However, Chromatographie
examination revealed no esterified sialic acids, but
only trace quantities of orcinol-positive material
with the same RF as free NANA were shown to be
released from tumor cells in the solid form. Since
mincing causes considerable cell disruption it is
probable that at least some of the sialic acid re
leased from the solid tumors is of intracellular
origin as distinct from a location in the peripheral
zone. Kimura et al. (12) have found no differences
in the total hexosamine and sialic acid content of
several types of murine tumor cells in both the
ascitic and solid form. Because lipide-bound sialic
acids are not always susceptible to neuraminidase,
the nature or accessibility of the linkage of the
sialic acid in the two forms of tumor cell may also
play a role (13).
We agree with Lasnitzki (14) that the differ
ences between cells from the solid and ascitic forms
of Sarcoma 37 appear to be due to a change in the
cells present rather than a selective process, be
cause we have found no evidence of bimodal dis
tribution in the rates of migration of the cells
during electrophoresis. The changes observed by
us are reversible, as evidenced by the fact that
cells from solid tumors revert to the original
ascitic form as assessed by the parameters used
here. If the comparison between 7-day Sarcoma
37 ascites and solid tumor cells is a valid one, we
may have described some changes associated with
a modulation (26) or a reversible transformation
in some of the properties of the peripheral zone
of mammalian cells.
Purdom, Ambrose, and Klein (17) also observed
a change in electrophoretic mobility when solid
tumors were transformed into ascites tumors.
However, these workers do not comment on the
early reversibility of the cellular electrokinetic
properties and were apparently unable, with their
data, to distinguish between changes due to modu
lation and those due to selection.
ACKNOWLEDGMENTS
One of us (G.M.W.C.) wishes to thank the Medical Re
search Council for financial assistance. We are also grateful to
Miss C. A. Cavanagh and Miss J. Davey for technical assist-
1817
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Physicochemical Differences between Ascitic and Solid Forms
of Sarcoma 37 Cells
G. M. W. Cook, G. V. F. Seaman and L. Weiss
Cancer Res 1963;23:1813-1818.
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